Long Interspersed Element-1 (LINE-1 or L1) is an abundant mobile genetic element that comprises approximately 17% of human DNA. Active human L1s are about 6 kb in length and they encode two proteins (ORF1p and ORF2p) that are critical for retrotransposition. Ongoing LINE-1 retrotransposition events contribute to inter-individual genetic variation, and, on occasion, LINE-1 insertions into genes can result in genetic disease. During the past decade, my laboratory has developed efficient assays to monitor LINE-1 retrotransposition in cultured mammalian cells. Here, we will use these assays in conjunction with genetic, molecular, and biochemical approaches to further elucidate the mechanism of L1 retrotransposition. We also will identify host proteins that affect LINE-1 retrotransposition in human cells. A basic mechanistic understanding of the process of L1 retrotransposition will lead to greater insight about how transposable element activity contributes to human disease, human genome evolution, and human diversity. PUBLIC HEALTH RELEVANCE: Long INterspersed Element-1 (LINE-1) is an abundant mobile genetic element that comprises 17% of human DNA. Active LINE-1 elements are able to retrotranspose or jump, inserting themselves into a new genomic location by a copy and paste mechanism. On occasion, new LINE-1 insertions in the germ line, in early development, or in somatic cells can result in disease-producing mutations. Despite the mutagenic potential of LINE-1 elements, little is known about the molecular mechanism of LINE-1 retrotransposition and even less is known about cellular proteins that influence this process. During the past decade, my laboratory has developed a toolbox to study LINE- 1 retrotransposition in a controlled manner in mammalian cultured cells. These tools have allowed significant progress in this field by my lab and others. In this proposal, we will use genetic, molecular biological, and biochemical approaches to further elucidate the molecular mechanism of LINE-1 retrotransposition. We also will explore how mutations in host proteins involved in host defense and/or DNA repair pathways affect LINE-1 jumping. Through these studies, we will learn more about how this intriguing family of repetitive DNA sequences contributes to the structure and function of the human genome.